We have previously reported that inactivation of c-kit and stem cell factor (SCF) might reduce interstitial Cajal-like cells (ICLCs) density, leading to gallbladder motility impairment and cholesterol gallstone (CG) formation. Based on bioinformatics prediction, this study explores the possible role of POU class 5 homeobox 1 (POU5F1) in c-kit/SCF regulation and investigates their function in ICLC activity and CG development. POU5F1 was identified as a transcription factor targeting both c-kit and SCF for transcription activation. They were poorly expressed in mice fed a lithogenic diet (LD) and mouse ICLCs treated with cholesterol. Upregulation of POU5F1 alleviated ICLC apoptosis, contraction dysfunction, and CG formation in the gallbladder wall of mice. Similarly, the POU5F1 upregulation enhanced the viability of ICLCs in vitro while reducing cell apoptosis. However, these effects were blocked by either c-kit or SCF knockdown. Furthermore, DNA methyltransferase 1 (DNMT1) and DNMT3B were identified as two important regulators suppressing POU5F1 transcription through DNA methylation. Knockdown of either DNMT1 or DNMT3B restored POU5F1 and c-kit/SCF levels, therefore reducing ICLC apoptosis and CG formation. In conclusion, this study demonstrates that DNMT1/DNMT3B-mediated DNA methylation of POU5F1 induces c-kit/SCF downregulation, thus promoting apoptosis of ICLCs and CG formation.